Impact of A90P, F106L and H64V mutations on neuroglobin stability and ligand binding kinetics.

Human neuroglobin (Ngb) is a hexacoordinated globin which binds some small ligands. Its function is still not well-established, even though Ngb seems to be implicated in the protection against neurodegenerative diseases. It has been shown by molecular dynamics and crystallography that ligand binding could occur thanks to a haem sliding mechanism specific to Ngb. In this paper, we studied some regions which could participate in this mechanism. We used UV-visible spectroscopy, CD and NMR to have a look on the protein structure and NMR and stopped-flow to study the ligand binding properties of the proteins. In the haem environment we mutated the distal histidine H64, the alanine A90 which is on the proximal F helix and the phenylalanine F106 which is close to the haem. We showed that both H64V and A90P variants, which affect the haem coordination, seemed to be important to haem and protein secondary structure stabilities whereas F106L mutation did not affect those properties. Then we confirmed that the cyanide binding kinetics were isomer dependent on wild-type Ngb and A90P and F106L variants. H64V Ngb variant had a behavior similar to wild-type Mb or Hb with a loss of the haem kinetic differentiation. Moreover, our results suggested that one haem isomer was more sensitive to A90P and F106L mutations. Those results brought some evidence that the haem sliding mechanism could occur for the cyanide binding and could be haem isomer dependent. The isomer forms may play distinct roles for the potential function of Ngb in vivo.

The dynamics of the non-heme iron in bacterial reaction centers from Rhodobacter sphaeroides.

We investigate the dynamical properties of the non-heme iron (NHFe) in His-tagged photosynthetic bacterial reaction centers (RCs) isolated from Rhodobacter (Rb.) sphaeroides. Mössbauer spectroscopy and nuclear inelastic scattering of synchrotron radiation (NIS) were applied to monitor the arrangement and flexibility of the NHFe binding site. In His-tagged RCs, NHFe was stabilized only in a high spin ferrous state. Its hyperfine parameters (IS=1.06±0.01mm/s and QS=2.12±0.01mm/s), and Debye temperature (θ(D0)~167K) are comparable to those detected for the high spin state of NHFe in non-His-tagged RCs. For the first time, pure vibrational modes characteristic of NHFe in a high spin ferrous state are revealed. The vibrational density of states (DOS) shows some maxima between 22 and 33meV, 33 and 42meV, and 53 and 60meV and a very sharp one at 44.5meV. In addition, we observe a large contribution of vibrational modes at low energies. This iron atom is directly connected to the protein matrix via all its ligands, and it is therefore extremely sensitive to the collective motions of the RC protein core. A comparison of the DOS spectra of His-tagged and non-His-tagged RCs from Rb. sphaeroides shows that in the latter case the spectrum was overlapped by the vibrations of the heme iron of residual cytochrome c(2), and a low spin state of NHFe in addition to its high spin one. This enabled us to pin-point vibrations characteristic for the low spin state of NHFe.

Chromosomal abnormalities in couples undergoing intracytoplasmic sperm injection. A study of 370 couples and review of the literature.

Intracytoplasmic sperm injection (ICSI) is now widely acknowledged as the most effective therapeutic approach to severe male infertility or unsuccessful in vitro fertilization. Cytogenetic investigations were performed in 370 females and 335 males prior to ICSI between January 1997 and April 2003. Nine men (2.7%) and 48 women (13%) had an abnormal karyotype, 44 females having some degree of numerical sex chromosome mosaicism. A review of the literature showed the prevalence of all types of chromosomal abnormalities to be much higher among male and female partners of couples examined prior to ICSI than among newborns. As most ICSIs are performed with ejaculated spermatozoa from oligospermic men, the distribution and the prevalence of the several types of chromosomal abnormalities are closer to those of oligospermic rather than azoospermic males. Our results combined with those of the literature stress the importance of karyotyping both male and female partners before ICSI is started. Adequate genetic counselling, possibly followed by prenatal diagnosis, should be offered if a chromosomal anomaly is detected.

Sex chromosome mosaicism in couples undergoing intracytoplasmic sperm injection.

BACKGROUND: Several studies have shown an increased frequency of constitutional chromosome aberrations in male and female partners of couples examined prior to ICSI. We conducted a cohort study to determine whether there was an increase in numerical sex chromosome mosaicism among couples undergoing ICSI compared with fertile couples. METHODS: Cytogenetic investigations were performed in 228 females and 208 males seen for ICSI between January 1997 and March 2001. They were matched to control females and males. RESULTS: Sex chromosome loss or gain was observed in at least one cell from 24.1% of ICSI women in comparison with 22% of controls (not significant). A significant difference between these two groups was found when X chromosome loss in at least two cells was considered, 9.6% for ICSI females versus 4.8% for controls (P = 0.01). No significant difference was observed between male groups concerning loss or gain of the X or Y chromosome. CONCLUSION: Our results support previously published studies indicating that the loss of an X chromosome in a single cell in females undergoing ICSI is probably an artefact. However, they suggest that a woman could have true sex chromosome mosaicism when two 45,X0 cells are found.

Critical assessment of the presence of an NADPH binding site on neutrophil cytochrome b558 by photoaffinity and immunochemical labeling.

The presumed NADPH dehydrogenase function of the heterodimeric cytochrome b558 in the neutrophil oxidase complex has been investigated by combined photoaffinity labeling and immunoblot analysis of membrane proteins from bovine neutrophils. The photoaffinity probe was a radiolabeled analog of NADPH, [4-[N-(4-azido-2-nitrophenyl)[3H]amino]butyryl]NADPH ([3H]azido-NADPH), and the antibodies were directed against the C-terminal regions of the two subunits of cytochrome b558. Plasma membrane vesicles obtained by differential centrifugation of bovine neutrophil homogenates were routinely used as a source of NADPH oxidase. They were permeabilized by sodium deoxycholate to facilitate the access of NADPH or its azido analog to the totality of the specific binding sites. In the absence of light, azido-NADPH behaved as a competitive inhibitor of NADPH oxidase with a Ki of 6 microM, and was able to bind to high-affinity specific binding sites with a Kd of 5-6 microM, indicating a higher affinity of the oxidase for the photoprobe than for the substrate NADPH (KM = 30-40 microM). Upon photolabeling, the oxidase was fully inactivated. Following resolution of the membrane proteins by SDS-PAGE, a predominant photolabeled protein band of 80-100 kDa was revealed, which coincided with the large subunit (beta) of cytochrome b558 identified by immunoblot in a parallel gel. The enzymatic deglycosylation of photolabeled neutrophil membranes shifted the masses of both the photolabeled band and the immunoreactive beta subunit from 80-100 to 55-65 kDa in accordance with the glycoprotein nature of the beta subunit.(ABSTRACT TRUNCATED AT 250 WORDS)